Imaging Dynamics and Interactions of Developmental Lineages in the Early Embryo

 描述（由申请人提供）：在发育过程中，脊椎动物中枢神经系统是通过将增殖的原始细胞群划分为区室并逐渐将这些细胞群细化为不同的功能亚区而形成的。在推测的中脑和后脑中，这些区室是由清晰的边界界面限制的谱系，确保来自这些区域的细胞不会混合，并且它们接收区域特异性信号以分别正确地发育成顶盖和小脑。这些区域特异性信号来源于位于中脑-后脑边界（MHB）的组织者，即峡部组织者。虽然峡部或MHB组织者的许多关键调控信号是已知的，但我们目前对这些区域如何启动、形成和维持的理解仍然很差。此外，这些区域和脑干的发育缺陷与智力残疾、自闭症和基亚里畸形等疾病有关。详细研究人类大脑在子宫内的形成以更好地了解其发育和发育障碍在伦理上是有问题的，但MHB是脊椎动物大脑进化上保守的三个组织者之一。因此，了解MHB如何在斑马鱼等模式物种中形成，将可能为人类大脑发育提供有价值的见解，特别是顶盖和小脑。然而，描述早期斑马鱼胚胎发育谱系动态和相互作用的技术能力和方法还有待证明。该项目将开发技术和试剂，以解决表征斑马鱼中脑和后脑中不同发育谱系的动态和相互作用的基本挑战，并可能扩展到其他功能区域和发育阶段，并可能扩展到其他模式物种。 MHB在斑马鱼中形成于细胞快速运动通过3D空间的早期发育阶段。在这个早期发育阶段，细胞和它们的身份在形态上是模糊的 并且只能通过基因表达来区分。因此，一个基本的挑战，在表征动力学和相互作用的推定中脑和后脑细胞MHB形成的能力，以获得体积图像在发育胚胎足够快，足够高的空间分辨率，以解决单细胞和识别成像细胞的关键调控基因的表达。该项目的目标1是产生具有荧光报告基因的转基因斑马鱼，其可信地标记未来的中脑和后脑细胞。本计画之目的二是发展一种以超短光脉冲为基础的新型光片显微术。延时，多分子光片显微镜将被用来测试两个主要的假设：最初重叠的边界中脑和后脑细胞是尖锐的通过细胞分选; 并且MHB的谱系限制性质与边界界面的锐化一致地建立。

 DESCRIPTION (provided by applicant): During development, the vertebrate central nervous system is formed by partitioning proliferating primordial cell populations into compartments and gradually refining these populations into distinct functional sub- regions. In the presumptive midbrain and hindbrain, these compartments are lineage restricted by a sharp boundary interface ensuring that cells from these regions do not mix and that they receive region specific signals to properly develop into the tectum and cerebellum, respectively. These region specific signals originate from an organizer, the isthmic organizer, positioned at the midbrain-hindbrain boundary (MHB). Though many of the key regulatory signals of the isthmic or MHB organizer are known, our current understanding of how these regions are initiated, formed, and maintained remains poor. Furthermore, developmental defects of these regions and brain stem have been linked to conditions such as intellectual disabilities, autism, and Chiari malformation. A detailed study of human brain formation in utero to better understand its development and developmental disorders is problematic ethically, but the MHB is one of three evolutionarily conserved organizers of the vertebrate brain. Thus, understanding how the MHB forms in model species such as zebra fish will likely provide valuable insights into human brain development, particularly the tectum and cerebellum. How- ever, the technological capability and methodology to characterize the dynamics and interactions of developmental lineages in the early zebra fish embryo has yet to be demonstrated. This project will develop the technologies and reagents that address fundamental challenges to characterizing the dynamics and interactions of distinct developmental lineages in the zebra fish midbrain and hindbrain and that may be extended to other functional regions and developmental stages and, potentially, other model species. The MHB forms in the zebra fish at an early developmental stage of rapid cell movement through 3D space. At this early developmental stage, cells and their identities are morphologically indistinct and may only be differentiated by gene expression. Thus, a fundamental challenge in characterizing dynamics and interactions of presumptive midbrain and hindbrain cells in MHB formation has been the capability to acquire volumetric images in developing embryos fast enough and with high enough spatial resolution to resolve single cells and to identify the imaged cells by the expression of key regulatory genes. Aim 1 of this project generates transgenic zebra fish with fluorescent reporters that fiducially mark future midbrain and hindbrain cells. Aim 2 of this project develops novel light sheet microscopy based on ultra-short optical pulses. Time-lapse, multimolecular light sheet microscopy will be used to test two main hypotheses: the initially overlapping boundary of mid- brain and hindbrain cells is sharpened through cell sorting; and lineage restriction properties of the MHB are established coincidentally with sharpening of the boundary interface.